Lipid droplet formation driven by PPARδ-mediated energy metabolism facilitates C2C12 myoblast differentiation

Skeletal muscle differentiation is an energy-intensive process that is tightly coupled to intracellular metabolic shifts. Lipid droplets (LDs), once considered inert lipid stores, are now recognized as dynamic organelles that integrate lipid metabolism, energy supply, and cell fate determination. In this study, we examined whether LD formation occurs under standard differentiation conditions and evaluated whether thermal stimulation (39°C) alters this process in C2C12 myoblasts. At 37°C, LD abundance increased transiently during early differentiation and was further enhanced by 39°C, coinciding with the upregulation of the LD-associated gene Plin2 . Knockdown of Plin2 reduced LD abundance and impaired myotube formation. Ppard expression rose under standard culture conditions and was further increased by thermal stimulation. Pharmacological activation of PPARδ with the agonist GW501516 elevated Plin2 expression, LD formation, and myogenic differentiation, whereas inhibition with the antagonist GSK3787 suppressed these thermal-induced effects. Glucose uptake also increased in a PPARδ-dependent manner under both conditions, with thermal stimulation specifically inducing Slc2a4 expression. Higher glucose availability accelerated LD formation and promoted myogenic differentiation, even in serum-free medium under thermal stimulation. Despite increased glucose uptake and LD formation, mitochondrial mass, ATP levels, and oxidative metabolism remained largely unchanged, indicating that glucose flux was redirected toward lipid synthesis. Overall, these findings identify PPARδ-mediated LD formation as a key metabolic adaptation facilitates myogenic differentiation and highlight thermal stimulation as a physiological enhancer, providing new insights into the metabolic regulation of muscle development and regeneration.